Luana Lamura

Mesenchymal stem cell‐derived interleukin‐6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012.

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells.

Increasing evidence suggests that bone marrow‐derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor‐associated neo‐angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF‐α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF‐α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF‐α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF‐α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin‐2, granulocyte‐colony stimulating factor, hepatocyte growth factor, interleukin (IL)‐6, IL‐8, and platelet‐derived growth factor‐BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF‐α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF‐α increased the ability of MSCs to induce migration of MCF‐7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo‐angiogenesis and tumor cell migration. J. Cell. Physiol. 226: 2131–2138, 2011.

Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells

Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27kip1 and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27kip1, with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27kip1 mRNA and in the nuclear levels of the p27kip1 transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27kip1 mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27kip1 mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27kip1 expression.

Expression and functional role of CRIPTO-1 in cutaneous melanoma

Background:CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma.Methods:Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers.Results:Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth.Conclusion:These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

Prognostic Applications of Gene Expression Signatures in Breast Cancer

Analysis by DNA microarrays has led to the identification of molecular subtypes of breast carcinomas that show a distinct expression profile. Several studies have demonstrated that this ‘intrinsic subtype’ classification has a strong prognostic value. In addition, gene expression profiling techniques have been used to identify gene signatures that could be associated with the outcome of breast cancer patients. Several different genomic tests have been shown to better define the prognosis of early-stage breast cancer patients as compared with conventional clinical and pathological characteristics of the tumors, and some assays are already commercially available. However, it must be emphasized that the prognostic power of these genetic classifiers has not been confirmed yet in prospective trials. Genetic signatures that might predict the activity of specific chemotherapy agents have also been developed by using gene expression profiling techniques. The same approach has been used to identify gene signatures associated with the activation of oncogenic pathways that might represent targets for molecular therapy of breast cancer. By using these approaches, gene expression techniques might significantly improve our ability to predict the risk of recurrence and to tailor the treatment for each individual breast cancer patient.

Pharmacokinetic evaluation of zoledronic acid

Introduction: Increased bone resorption is associated with several diseases, including osteoporosis, bone metastases and Pagets disease of bone. Zoledronic acid (ZA) is the most potent of the clinically available bisphosphonates. In addition to its antiresorptive activity, there has been increasing evidence to suggest that it also has anticancer properties. Areas covered: This article is complied through PubMed and Medline databases searches on ZA. In this review, the authors summarize the current knowledge (up to December 2010) on the pharmacodynamic and pharmacokinetic properties of ZA. Expert opinion: ZA is a well-tolerated and effective drug in the management of metabolic as well as cancer-related bone disease. Clinical benefits in cancer patients include improvement in bone pain, reduction in skeletal events and delay of time to first skeletal event. However, novel indications for this drug are emerging from clinical studies in early breast cancer. Recent findings suggest that the addition of ZA to endocrine therapy can significantly prevent bone loss in premenopausal patients. Increasing evidence also indicates a potential anticancer activity of ZA, although this property needs to be further explored.

Anticancer effect of bisphosphonates: new insights from clinical trials and preclinical evidence.

Bisphosphonates (BPs) are cornerstones in the treatment of patients with compromised skeletal integrity (either cancer related or not). However, a major indication for BPs use remains the treatment of patients with advanced cancer metastatic to the bone. Recently, several observations derived from clinical trials, primarily aimed at establishing the impact of BPs on the bone health of cancer patients, suggested a potential role for these agents as direct anti-tumor drugs. Consequently, a series of preclinical works were produced with the aim of clarifying the mechanism underlying this observed effect. However, the impact of such data is still under debate owing to the intrinsic weakness of observations from trials not adequately powered to support them. In conclusion, the entire matter remains one of the most intriguing in oncology, and data from ongoing and planned future studies will surely provide us with more information on the great potential of BPs in the adjuvant setting.

Abstract 565: Effects of zoledronic acid on the interaction between mesenchymal stem cells and breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Zoledronic acid (ZA) is the mainstay of treatment of bone metastases associated with a wide variety of tumors, including breast cancer. It has been recently shown that ZA increases the progression free-survival of estrogen receptor (ER)-positive breast cancer patients by reducing both loco-regional and distant metastases, suggesting that this compound might act directly on breast cancer cells. However, ZA very rapidly concentrates in the bone following intravenous administration, and therefore this drug is unlikely to have a direct effect on cell populations outside the bone. Recent reports have shown that bone-marrow-derived mesenchymal stem cell (MSCs) are recruited to the stroma of developing tumors where they increase the metastatic potential of breast cancer cells by secreting cytokines, chemokines and growth factors that sustain breast cancer migration, invasion and metastasis. In particular, the chemokine CCL5 (RANTES) plays an important role in this phenomenon. Therefore, we investigated the effects of ZA on the ability of primary MSCs to secrete cytokines and growth factors that might be involved in breast cancer progression. We found that treatment with ZA produced marginal effects on the growth of human primary MSCs with an approximately 25% growth inhibition at 20 μM. In contrast, treatment with similar concentrations of ZA almost completely suppressed the ability of MSCs to secrete RANTES (>90% reduction). The effect of ZA on RANTES was quite specific, since marginal inhibition of the secretion of different angiogenic growth factors, such as VEGF, IL-8 and bFGF was observed. Treatment of MSCs with ZA also produced a 50% reduction in the levels of secretion of IL-6 that has been previously shown to act as a potent paracrine growth factor for human breast cancer cells. Both RANTES and IL-6 transcripts were significantly reduced in MSCs following exposure to ZA, suggesting a transcriptional regulation of these cytokines by ZA. Conditioned medium from ZA-treated MSCs showed a reduced ability to promote the migration of ER-positive MCF-7 breast cancer cells through a fibronectin-coated membrane as compared with conditioned medium from untreated cells. In addition, antibodies anti-RANTES and anti-IL-6, alone or in combination, produced an approximately 50% reduction of the migration of MCF-7 induced by conditioned medium from untreated MSCs. In co-culture assays, treatment with ZA reduced the ability of MSCs to sustain the growth of breast cancer cells. Finally, the migration of MSCs was reduced by approximately 45% following treatment with ZA. Taken together, these data suggest that ZA might exert its antitumor activity in the bone marrow microenvironment by inhibiting the migration of MSCs and by blocking the ability of MSCs to secrete factors involved in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 565.

Abstract 3581: Characterization of human breast cancer cells with acquired resistance to the EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The epidermal growth factor receptor (EGFR) and the ErbB-2 receptor are co-expressed in a subset of breast carcinomas that show a more aggressive phenotype. The dual EGFR/ErbB-2 tyrosine kinase inhibitor lapatinib has been recently approved for the treatment of metastatic breast cancer patients whose tumors overexpress ErbB-2. We investigated the mechanisms of acquired resistance to lapatinib in breast cancer cells. For this purpose, we used as model the SK-Br-3 human breast carcinoma cell line that express high levels of ErbB-2, and moderate levels of EGFR and ErbB-3. We selected lapatinib-resistant SK-Br-3 cells by stepwise dose escalation of lapatinib. After six months of selection, we isolated SK-Br-3 cells that were able to grow in 1 μM lapatinib. The IC50 of lapatinib for parental SK-Br-3 cells was 140 nM, whereas the IC50 for the lapatinib-resistant SK-Br-3 derived cell line (Lap-R) was > 4 μM. Treatment with lapatinib significantly inhibited the activation of EGFR, ErbB-2 and ErbB-3 in both parental and Lap-R SK-Br-3 cells. However, following treatment with lapatinib, Lap-R cells showed a persistent activation of AKT and MAPK, which in contrast were completely inhibited in parental SK-Br-3 cells, as measured by both western blotting and a Luminex-based phosphoprotein array. In addition, treatment with lapatinib produced a slight activation of c-Src in parental cells, whereas significant levels of c-Src activation were detected in Lap-R cells cultured in presence of lapatinib. Lap-R cells also showed higher levels of expression of the chemokine receptor CXCR-4 than parental cells. In agreement with these findings, Lap-R cells showed a significantly higher ability to invade a matrigel-coated membrane as compared with parental SK-Br-3 cells, as measured with a Boyden-chamber based colorimetric invasion assay. Preliminary findings demonstrate that inhibition of c-Src with the selective src kinase inhibitor saracatinib produces a more significant inhibition of Lap-R cell invasion as compared with parental SK-Br-3 cells. Taken together, these findings suggest that activation of c-Src signalling might play a role in acquired resistance to lapatinib in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3581. doi:10.1158/1538-7445.AM2011-3581